Biology subjects

Totanes, F. I. G.

Publications and source records attributed to Totanes, F. I. G..

5 recordsLinked to original sources

bioRxiv2025

Validated Antimalarial Drug Target Discovery using Genome-Scale Metabolic Modelling

Given the rapid resistance of Plasmodium falciparum to antimalarial drugs, there is a continual need for new treatments. A genome-scale metabolic (GSM) model was developed with integrated omic and constraint-based, experimental flux-balance data to predict genes essential for P. falciparum growth as drug targets. We selected the highly ranked P. falciparum UMP-CMP kinase (UCK) to test its necessity and the ability to inhibit growth with inhibitors. Conditional deletion mutants using the DiCre recombinase system, generated by CRISPR-Cas genome editing, exhibited defective asexual growth and stage-specific developmental arrest. Based on in silico and in vitro screening, inhibitors were identified that are selective for P. falciparum UCK and exhibit antiparasitic activity. This study, for the first time, shows assertions from a GSM model identifying novel, validated, "druggable" targets. These findings show a role for GSM models in antimalarial drug discovery and identify P. falciparum UCK as a novel, valid malaria drug target.

microbiology↗

bioRxiv2024

DNA replication dynamics are associated with genome composition in Plasmodium species

Plasmodium species have variable genome compositions: many have an A/T-content of at least 80% while others are similar in composition to human cells. Here, we made a direct comparison of DNA replication dynamics in two Plasmodium species whose genomes differ by [~]20% A/T-content. This yielded fundamental insights into how DNA composition may affect replication. The highly A/T-biased genome of P. falciparum showed unusual replication dynamics that were not observed in the more balanced P. knowlesi - which had dynamics more like those of human cell lines. We observed that replication forks moved 50% slower in P. falciparum than in P. knowlesi. In P. falciparum, replication forks slowed down over the course of S-phase whereas in P. knowlesi, fork speed increased as in human cells. Furthermore, in both P. knowlesi and human cells, replication forks were strikingly slowed by sequences of particularly high A/T-bias, but in P. falciparum, although replication forks were inherently slow, they were not particularly slow in such biased sequences. Thus, the replisome of P. falciparum may have evolved alongside its extremely biased genome, making it unusually robust to sequence bias. Since several antimalarial drugs act to stall DNA replication, this study may have implications for the effectiveness of, and development of, antimalarial therapies.

microbiology↗

bioRxiv2022

Replication origin mapping in the malaria parasite Plasmodium falciparum

The malaria parasite Plasmodium falciparum replicates via schizogony: a fundamentally unusual type of cell cycle involving asynchronous replication of multiple nuclei within the same cytoplasm. It also has one of the most A/T-biased genomes ever sequenced. Here, we present the first comprehensive study of the specification and activation of DNA replication origins during Plasmodium schizogony. Potential replication origins were found to be abundant, with ORC1-binding sites detected every [~]800 bp throughout the genome. They had no motif enrichment, but were biased towards areas of higher G/C content. Origin activation was then measured at single-molecule resolution via new DNAscent technology that measures fork movement by detecting base analogues BrdU and EdU in DNA sequenced on the Oxford nanopore platform (https://github.com/MBoemo/DNAscent). DNAscent-called origins were found to be much less dense than ORC1-binding sites, with origins activated preferentially in areas of low transcriptional activity. Consistently, replication forks moved fastest through the most lowly transcribed genes, suggesting that conflicts between transcription and origin firing inhibit efficient replication, and that P. falciparum has evolved its S-phase to minimise such conflicts.

microbiology↗

bioRxiv2021

Dynamics of DNA replication during male gametogenesis in the malaria parasite Plasmodium falciparum

Malaria parasites undergo a single phase of sexual reproduction in their complex lifecycle, during which they cycle between mosquito and vertebrate hosts. Sexual reproduction occurs only at the point when parasites move into the mosquito host. It involves specialised, sexually committed cells called gametocytes, which develop very rapidly into mature gametes and then mate inside the mosquito midgut. The gamete development process is unique, involving unprecedentedly fast replication and cell division to produce male gametes. A single male gametocyte replicates its [~]23Mb genome three times over to produce 8 genomes, segregates these into newly-assembled flagellated gamete cells and releases them to seek out female gametes, all within [~]15 minutes. Here, for the first time, we use fluorescent labelling of de novo DNA synthesis to follow this process at the whole-cell and single-molecule levels, yielding several novel observations. Firstly, we confirm that no DNA replication occurs before gametogenesis is triggered, although the origin recognition complex protein Orc1 is abundant even in immature gametocytes. Secondly, between repeated rounds of DNA replication there is no detectable karyokinesis - in contrast to the repeated replicative rounds that occur in asexual schizonts. Thirdly, cytokinesis is clearly uncoupled from DNA replication, and can occur even if replication fails, implying a lack of cell cycle checkpoints. Finally the single-molecule dynamics of DNA replication are entirely different from those in asexual schizonts.

microbiology↗

bioRxiv2021

Plasmodium falciparum GBP2 is a telomere-associated protein that binds to G-quadruplex DNA and RNA

In the early-diverging protozoan parasite Plasmodium, few telomere-binding proteins have been identified and several are unique. Plasmodium telomeres, like those of most eukaryotes, contain guanine-rich repeats that can form G-quadruplex structures. In model systems, quadruplex-binding drugs can disrupt telomere maintenance and some quadruplex-binding drugs are potent anti-plasmodial agents. Therefore, telomere-interacting and quadruplex-interacting proteins may offer new targets for anti-malarial therapy. Here, we report that P. falciparum GBP2 is such a protein. It was identified via Proteomics of Isolated Chromatin fragments, applied here for the first time in Plasmodium. In vitro, PfGBP2 binds specifically to G-rich telomere repeats in quadruplex form and it can also bind to G-rich RNA. In vivo, PfGBP2 partially colocalises with the known telomeric protein HP1 but is also found in the cytoplasm, probably due to its affinity for RNA. Consistently, its interactome includes numerous RNA-associated proteins. PfGBP2 is evidently a multifunctional DNA/RNA-binding factor in Plasmodium.

microbiology↗